The current research of our group is focused on the mechanism of action of ATP-dependent molecular chaperones and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones are present in all organisms and are highly conserved. They interact with proteins to mediate protein remodeling, folding, unfolding, assembly, and disassembly without themselves being part of the final complex. Chaperones participate in many cellular processes such as DNA replication, regulation of gene expression, cell division, membrane translocation, and protein degradation. Many are induced by environmental stresses and play a critical role during cell stress by preventing the appearance of folding intermediates that lead to irreversibly damaged proteins. They assist in the recovery from stress by refolding and reactivating damaged proteins and by disaggregating aggregated proteins. Some chaperones interact with specific proteolytic components forming compartmentalized ATP-dependent proteases. When associated with proteases, chaperones function in the delivery of damaged proteins and specific regulatory proteins to the proteolytic component. We previously discovered that Escherichia coli ClpA, an AAA+ ATPase and the regulatory component of ClpAP protease, has molecular chaperone activity. This finding demonstrated that Clp ATPases comprise a family of ATP-dependent chaperones and that molecular chaperones participate directly in proteolysis. One of the aims of our group is to elucidate the mechanism of action of Clp ATPases, including ClpA and ClpX, and their role in proteolysis in combination with ClpP, the proteolytic component of the ClpAP and ClpXP proteases. To examine the mechanism of interaction of ClpAP with dimeric substrates, single-round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in fundamentally different ways. We are also studying the mechanism of protein remodeling by the DnaK/Hsp70 chaperone system of E. coli. The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogues in E. coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK, and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. However, CbpA differs from DnaJ in some aspects. For example, it lacks autonomous chaperone activity. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of T. thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. We isolated E. coli YccD because of its potential functional relationship to CbpA and discovered that the purified protein interacted with CbpA. The consequence of this interaction was the inhibition of both the DnaJ-like co-chaperone activity and the DNA binding activity of CbpA. Based on these results, the product of the yccD gene has been named CbpM, for "CbpA Modulator". Together CbpA and CbpM are capable of activating and modulating the activity of the DnaK chaperone system, respectively. We previously showed that plasmid P1 RepA is a substrate for both ClpA and DnaK/DnaJ/GrpE chaperone action. These molecular chaperones convert inactive RepA dimers into monomers that are active in P1 ori DNA binding. To gain insight into the mechanism of monomerization, we made a molecular model of RepA and tested the predictions of the model. By using fold-recognition programs we found that RepA shares structural homology with RepE of F plasmid, although the sequence of RepA is not homologous to that of RepE or other plasmid initiator proteins. From the alignment we built a model based on the RepE crystal structure and constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. The results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.